Tatjana S. Kostic

Inhibition of Rat Testicular Androgenesis by a Polychlorinated Biphenyl Mixture Aroclor 1248

Abstract Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin–stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Δ4-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/lyase, and 17β-hydroxysteroid dehydrogenase activities.

Effects of acute and chronic immobilization stress on rat Leydig cell steroidogenesis

In rats, acute immobilization (IMO) stress (2 h) induced a fall in the serum androgen concentrations (T+DHT) without detectable changes in serum luteinizing hormone (LH) values. In vitro studies, using a suspension of Leydig cells from adult rat testis, demonstrated that acute stress inhibited conversion of progesterone (P) or 17hydroxyprogesterone (17OHP) to T while conversion of androstendione (delta 4 A) was not affected. Acute IMO reduced activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and decreased basal and hCG-stimulated progesterone and androgen production. Chronic IMO stress (2 h daily for 10 days) induced a decrease in serum androgen level with decline in serum LH values. In vitro, hCG-stimulated progesterone and androgen production by suspension of Leydig cells, as well as conversion of P and 17OHP to T were not significantly altered. Our data demonstrates that acute IMO stress impaired testicular steroidogenesis primarily at the testicular level (decreasing the activity of certain enzymes), while chronic IMO stress exerts the effect mainly on the hypothalamic-pituitary axis; reduced serum LH levels elicit a decrease in serum androgen levels.

Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis

The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2x2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N(omega)-nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2x600 microg/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes.

The involvement of nitric oxide in stress-impaired testicular steroidogenesis

The participation of the nitric oxide (NO) pathway in downregulation of testicular steroidogenesis in normal and stressed rats was investigated both in vivo and in vitro. In Leydig cells from normal animals, isosorbide dinitrate, an NO donor, decreased the human chorionic gonadotropin (CG)-stimulated and progesterone-derived androgen production. Also, the intratesticular injection of a precursor of NO, arginine (10 mg/testis), transiently decreased serum androgen levels and inhibited human CG-stimulated androgen production in acute testicular cultures. These effects were eliminated in rats cotreated with Nomega-nitro-L-arginine methyl ester (L-NAME) (2 X 600 microg/each testis). Acute immobilization stress (2 h) decreased serum androgen levels and inhibited human CG-stimulated androgen production in vitro. These effects were accompanied by a significant increase in nitrite, a stable oxidation product of NO, in testicular cultures. Bilateral intratesticular injection of L-NAME prevented the stress-induced decrease of human CG-stimulated androgen production, and significantly reduced the nitrite levels. These results implicate NO in normal and stress-impaired testicular steroidogenesis.

Testosterone-Induced Modulation of Nitric Oxide-cGMP Signaling Pathway and Androgenesis in the Rat Leydig Cells

Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.

The effect of opioid antagonists in local regulation of testicular response to acute stress in adult rats

The present study examined the effects of naloxone (N) and naltrexone-methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) on testicular steroidogenesis during acute immobilization stress (IMO; 2 h) in adult rats. Unstressed rats as well as IMO rats were treated by unilateral intratesticular injection of N (20 micrograms/testis), NMB (36 micrograms/testis), or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats serum T levels were significantly reduced, while serum luteinizing hormone levels were not affected. N and NMB normalized serum T levels in IMO rats and had no effects in controls. In IMO rats the activities of 3 beta-hydroxysteroid dehydrogenase (HSD) and P450(17 alpha, lyase) were significantly reduced, while the activity of 17 beta-HSD was not affected. N and NMB antagonized the inhibitory effect of IMO on 3 beta-HSD and P450(17 alpha, lyase) but did not alter enzyme activity in freely moving rats. Acute IMO decreased basal and human chorionic gonadotropin-stimulated androgen production by hemitestis preparation, but N (10(-4) M) added directly to the incubation medium blocked the decrease and had no effect on testes from freely moving control rats. These results support the conclusion that endogenous opioid peptides are potentially important paracrine regulators of testicular steroidogenesis under stress conditions.

Pharmacological Doses of Testosterone Upregulated Androgen Receptor and 3-Beta-Hydroxysteroid Dehydrogenase/Delta-5-Delta-4 Isomerase and Impaired Leydig Cells Steroidogenesis in Adult Rats

Anabolic androgenic steroids (AAS) are testosterone derivatives originally designed to enhance muscular mass and used for the treatment of many clinical conditions as well as in contraception. Despite popular interest and abuse, we still lack a broad understanding of effects of AAS on synthesis of steroid hormones on the molecular level. This study was designed to systematically analyze the effects of pharmacological/high doses of testosterone on steroidogenic machinery in Leydig cells. Two different experimental approaches were used: (1) In vivo experiment on groups of adult male rats treated with testosterone for 1 day, 2 weeks, and 2 months; (2) Direct in vitro testosterone treatment of Leydig cells isolated from intact rats. Result showed that prolonged in vivo treatment with testosterone decreased the expression of Scarb1 (scavenger receptor class B type 1), Tspo (translocator protein), Star (steroidogenic acute regulatory protein), Cyp11a1 (cholesterol side-chain cleavage enzyme), and Cyp17a1 (17α-hydroxylase/17, 20 lyase) in Leydig cells. Oppositely, the expression of Hsd3b (3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase), Ar (androgen receptor), and Pde4a/b (cyclic adenosine monophosphate-dependent phosphodiesterases) was increased. Androgenization for 2 weeks inhibited Cyp19 (aromatase) transcription, whereas 2-month exposure caused the opposite effect. Direct in vitro testosterone treatment also decreased the expression of Cyp11a1, Cyp17a1, and Cyp19a1, whereas Hsd3b was upregulated. The results of expression analysis were supported by declined steroidogenic capacity and activity of Leydig cells, although conversion of pregnenolone to progesterone was stimulated. The upregulation of AR and 3βHSD in testosterone-impaired Leydig cells steroidogenesis could be the possible mechanism that maintain and prevent loss of steroidogenic function.

Structural complexity of the testis and PKG I/StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

The adaptive response of adult rat Leydig cells to repeated immobilization stress: The role of protein kinase A and steroidogenic acute regulatory protein

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study, we investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Δ4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells

Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 microg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was significantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data confirmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition.